Though recurrent Clostridium difficile infections (CDI) are common and pose a major clinical concern, data are lacking regarding mortality among patients who survive their initial CDI and have subsequent recurrences. Risk factors for mortality in patients with recurrent CDI are largely unknown.

Methods

Veterans Affairs patients with a first CDI (positive C. difficile toxin(s) stool sample and ≥ 2 days CDI treatment) were included (2010–2014). Subsequent recurrences were defined as additional CDI episodes ≥ 14 days after the stool test date and within 30 days of end of treatment. A matched (1:4) case-control analysis was conducted using multivariable conditional logistic regression to identify predictors of all-cause mortality within 30 days of the first recurrence.

In our national cohort of Veterans, crude mortality decreased by 44% from the initial episode to the third recurrence. Treatment with antibiotics, PPIs, and underlying co-morbidities were important predictors of mortality in recurrent CDI. Our study assists healthcare providers in identifying patients at high risk of death after CDI recurrence.

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Objectives

This study aimed to examine changes to the microbiota composition and metabolic profiles of seven patients with recurrent Clostridium difficile infection (rCDI), following treatment with faecal microbiota transplant (FMT).

Summary

Objectives

This study aimed to examine changes to the microbiota composition and metabolic profiles of seven patients with recurrent Clostridium difficile infection (rCDI), following treatment with faecal microbiota transplant (FMT).

Methods

16S rDNA sequencing and 1H NMR were performed on faecal samples from the patients (pre-, post-FMT, and follow-up) and the associated donor samples. Sparse partial-least-square analysis was used to identify correlations between the two datasets.

Results

The patients’ microbiota post-FMT tended to shift towards the donor microbiota, specifically through proportional increases of Bacteroides, Blautia, and Ruminococcus, and proportional decreases of Enterococcus, Escherichia, and Klebsiella. However, although cured of infection, one patient, who suffers from chronic alcohol abuse, retained the compositional characteristics of the pre-FMT microbiota. Following FMT, increased levels of short-chain fatty acids, particularly butyrate and acetate, were observed in all patients. Sparse partial-least-square analysis confirmed a positive correlation between butyrate and Bacteroides, Blautia, and Ruminococcus, with a negative correlation between butyrate and Klebsiella and Enterococcus.

Conclusions

Clear differences were observed in the microbiota composition and metabolic profiles between donors and rCDI patients, which were largely resolved in patients following FMT. Increased levels of butyrate appear to be a factor associated with resolution of rCDI.

Introduction

Although Clostridium difficile is present in the intestines of ∼3–5% of healthy adults,1 the occurrence of C. difficile infection (CDI) in healthy individuals is relatively uncommon due to the protective effect of the gut microbiota. The incidents and severity of CDI has risen significantly over the last decade, and it is now recognised as the main causative agent of healthcare-associated infectious diarrhoea in hospitals worldwide.2 The standard treatment for CDI is the administration of metronidazole for mild to moderate infections, and oral vancomycin or fidaxomicin for severe infections and relapses. The ability of C. difficile to form spores, coupled with the increase in antibiotic-resistant strains, can lead to persistence of infection, relapses, and the administration of more antibiotics, which further depletes the commensal bacteria. This creates an environment that is more favourable to C. difficile, thus setting up a cycle of relapse and re-infection. It is estimated that 20-30% of patients who develop a first episode of CDI go on to have at least one relapse, and of these, a further 60% develop further episodes of relapses.3 This increases the need for further antibiotics, the risk of antibiotic-resistance in the gut commensal flora, and costs to the health service, with each episode of CDI estimated to cost approximately £7000 in 2010.4

Faecal microbiota transplants (FMT) represents an effective alternative to antibiotics to treat recurrent CDI (rCDI), with a primary cure rate as high as 91%.5 The central tenet behind FMT is that the introduction of a healthy bacterial community into the intestines produces an environment that is less favourable to C. difficile by increasing colonisation resistance and reinstating a protective effect. The advantages of this treatment are that it is quick, cost-effective, and could help to eradicate antibiotic resistant strains of C. difficile.

It is known that a dysbiotic gut microbiota increases the risk of developing CDI, however whether there is a common element within this community composition that could help to determine if a patient is at greater risk of rCDI is as yet unknown. The reduction in diversity within the dysbiotic gut microbiota would also suggest a reduction in metabolic potential through the loss of gene diversity. The functional redundancy6 within the gut microbiota suggests, that metabolic function is more relevant than which species are present or absent. Whilst a number of studies have looked at the changes in microbiota composition due to FMT,7,8,9,10 we know little about the changes to the metabolic capacities of the altered microbiota. The aim of this study was to assess FMT-induced changes in both the microbial community structure and metabolite profiles of the gut microbiomes of seven patients with rCDI, as well as those of their associated FMT donors.

Patients and methods

Patients

Patients were selected as candidates for the FMT procedure if they had at least two confirmed recurrences of CDI. C. difficile testing was based on a two stage algorithm in line with Public Health England recommendations.11 This involves screening faecal samples by glutamate dehydrogenase enzyme immunoassay (Techlab, USA), followed by C. difficile toxin testing by enzyme immunoassay (Techlab, USA). Glutamate dehydrogenase positive, toxin negative samples were further tested for the presence of toxigenic genes by PCR. FMT exclusion criteria included immunocompromised patients, those aged less than 16, and those with severe comorbidities which would make the patient unfit for endoscopy. FMT was introduced into clinical care at Norfolk and Norwich University Hospital following approval by the New Therapies committee, and was performed in accordance with the Helsinki Declaration of 1975. Patients were consented for the study by a clinician following a detailed discussion of the procedure with the patient or their next of kin. All patient data is fully anonymised.

Donor screening

The faecal donors used for the cohort of patients who underwent FMT in this study were both healthy Caucasian males with a BMI between 24 and 27 kg/m2, aged 36 (D05) and 30 (D03) years of age, respectively. Potential donors were asked to complete a questionnaire adapted from van Nood et al.12 regarding their medical history and lifestyle habits to identify risk factors for potentially transmittable diseases. Eligible candidates provided blood and stool samples for laboratory screening tests. Blood samples were screened for hepatitis A, B, C, and E antibodies, HIV 1 & 2, human T-lymphotropic virus 1 & 2, Epstein-Barr virus, Cytomegalovirus, syphilis, Entamoeba histolytica, Strongyloides stercoralis, and Treponema pallidum. Stool samples were tested for the presence of C. difficile or its toxins, Helicobacter pylori antigen, Norovirus, methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, extended-spectrum β-lactamase-producing organisms, carbapenemase-producing Enterobacteriaceae, Escherichia coli O157, Salmonella spp., Shigella spp., and Campylobacter species. In addition, microscopy was used to investigate for ova, cysts, and parasites. Prior to the donation of stool samples for each FMT procedure, donors were asked to refrain from eating peanuts and shellfish, and to complete a short screening questionnaire to confirm no changes to health or lifestyle since the last donor screening that may put the patient at risk.

Faecal suspension preparation

Donor faeces were collected in a sterile container on the day of the procedure, and transferred to a sterilised class II safety cabinet (Walker Ltd, UK). A maximum of 80 g of donor stool was homogenised with sterile saline (0.9%), to a ratio of 5 ml saline per gram of stool, in a strainer bag (BA6141/STR; Seward Limited, UK) using a Stomacher® 400 Circulator (Seward Limited, UK) set to 230 RPM for a duration of 1 min. The filtered faecal preparation was drawn up into labelled sterile 60 ml syringes using nasojejunal tubing connected to the Luer lock. The syringes were secured with sterile Luer lock caps and transported immediately to the hospital. Aliquots of the donor faecal sample were immediately stored at −20 °C until analysis.

Faecal suspension infusion

Patients were prescribed oral vancomycin 500 mg four times daily for 4 days, with the last dose received the night before the procedure. Also, on the day before the FMT procedure, a bowel lavage is performed using 4 l of macrogol solution (Klean-Prep, Norgine). Patients were taken to the endoscopy unit for insertion of nasojejunal tube the night before the procedure. Our FMT protocol was adapted from that of van Nood et al.12 On the day of FMT infusion, the patient’s headrest was elevated to 45°, patency of the nasojejunal tube was checked by flushing with water, and 420 ml of faecal suspension was delivered slowly by the patient’s bedside in the isolation room via a nasojejunal tube using the prefilled syringes. This was performed at a rate of ∼20 ml per minute with a break of 5-10 minutes applied halfway through the procedure. Post-infusion instructions were to monitor observations, and record bowel motions. Patients could take on fluids one hour after the procedure, and were observed overnight before discharge the next day at the earliest. Although there are no agreed durations of follow-up post-FMT,13 van Nood et al.12 used two endpoints to measure cure, namely no relapse after 5 weeks, and no relapse after 10 weeks. Resolution was defined as type 4 or less on the Bristol stool chart or stool normal for the patient e.g. in case of percutaneous endoscopic gastrostomy feeding. We followed patients up by telephone or in person if they were re-admitted into the hospital for an unrelated illness. Post-FMT samples were collected after a minimum of 10 days post-FMT, and postal kits were provided to patients who were willing to donate a ‘follow-up’ sample up to 2 weeks later.

Sample analysis

Faecal microbiota analysis

Faecal samples were collected from recipients within 9 days prior to FMT, however the pre-FMT sample for patient R13 was not collected within this timeframe, and a previously frozen sample obtained whilst the patient was suffering from the same episode of CDI was used. Further samples were collected for all recipients following the procedure (‘post-FMT’ range: 11–141 days; ‘follow-up’ range: 4–14 days after post-FMT sample), and stored at −20 °C until analysis. The DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals, UK) with a bead-beating step.14 DNA yield was quantified using the Qubit fluorometer prior to the samples being sent to the Earlham Institute (UK), where the V4 hypervariable region of the 16S rRNA genes were amplified using the 515F and 806R primers with built-in degeneracy.15 The amplicons were sequenced using paired-end Illumina sequencing (2 × 250 bp) on the MiSeq platform (Illumina, USA). Sequencing data, for the 21 samples that had an appropriate level of sequencing depth, were analysed using the Quantitative Insights Into Microbial Ecology (QIIME) 1.9 software and RDP classifier 16S rRNA gene sequence database.16,17 The trimmed reads were filtered for chimeric sequences using ChimeraSlayer, bacterial taxonomy assignment with a confidence value threshold of 50% was performed with the RDP classifier (version 2.10), and trimmed reads clustered into operational taxonomic units at 97% identity level. Alpha diversity and rarefaction plots were computed using the Chao1 index. Weighted and unweighted UniFrac distances were used to generate beta diversity principal coordinates analysis plots, which were visualised using the Emperor tool.

Faecal metabolite analysis

A known mass (∼ 100 mg) of thawed faecal samples were added to sterile tubes. The faecal waters were generated by adding the phosphate buffer (prepared in D2O) to 8.3% w/v. Homogenised faecal waters were centrifuged at 16,200 x g at room temperature for 5 min. The supernatants were filter sterilised (0.2 µm) and placed in a 5 mm NMR tube. The 1H NMR spectra were recorded at 600 MHz on a Bruker Avance spectrometer (Bruker BioSpin GmbH, Germany) running Topspin 2.0 software and fitted with a cryoprobe and a 60-slot autosampler. Each 1H NMR spectrum was acquired with 1280 scans, a spectral width of 12,300 Hz, and an acquisition time of 2.67 s. The “noesypr1d” pre-saturation sequence was used to suppress the residual water signal with a low-power selective irradiation at the water frequency during the recycle delay and a mixing time of 10 ms. Spectra were transformed with a 0.3 Hz line broadening, and were manually phased, baseline corrected, and referenced by setting the TSP methyl signal to 0 ppm. The metabolites were quantified using the software Chenomx® NMR Suite 7.0TM.

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Microbiome profiling through 16S rRNA gene sequencing has proven to be a valuable tool to characterize the diversity and composition of gut microbial communities, including in studies of CDI development and recurrence.8

Abstract

Microbiome profiling through 16S rRNA gene sequence analysis has proven to be a useful research tool in the study of C. difficile infection (CDI); however, CDI microbiome studies typically report results at the genus level or higher, thus precluding identification of this pathogen relative to other members of the gut microbiota.

Accurate identification of C. difficile relative to the overall gut microbiome may be useful in assessments of colonization in research studies or as a prognostic indicator for patients with CDI.

To investigate the burden of C. difficile at the species level relative to the overall gut microbiome, we applied a high-resolution method for 16S rRNA sequence assignment to previously published gut microbiome studies of CDI and other patient populations. We identified C. difficile in 131 of 156 index cases of CDI (average abundance 1.78%), and 18 of 211 healthy controls (average abundance 0.008%).

We further detected substantial levels of C. difficile in a subset of infants that persisted over the first two to 12 months of life. Correlation analysis of C. difficile burden compared to other detected species demonstrated consistent negative associations with C. scindens and multiple Blautia species.

These analyses contribute insight into the relative burden of C. difficile in the gut microbiome for multiple patient populations, and indicate that high-resolution 16S rRNA gene sequence analysis may prove useful in the development and evaluation of new therapies for CDI.

Introduction

Clostridium difficile infection (CDI) poses a major healthcare burden to the global population, with an estimated 450,000 cases and 29,000 deaths in the United States annually.1,2 CDI is often associated with antibiotic treatment and is frequently acquired by patients during hospitalization.

Multiple diagnostic tests for CDI are available and hospitals commonly use a combination of enzyme immunoassay (EIA) and glutamate dehydrogenase (GDH) testing in tandem with real-time polymerase chain reaction (PCR) for increased sensitivity and shorter turnaround time.3

After diagnosis, patients with CDI are typically treated with metronidazole and/or vancomycin depending on symptom severity.3 Treatment failure is estimated to occur in 20% of patients, resulting in a recurrent CDI population that may require other treatment strategies.4,5 The development of microbial-based therapeutics, such as fecal microbiota transplantation (FMT) and combinations of selected microbes for the treatment of recurrent CDI suggests that mixtures of commensal microbes may be routinely utilized in the future as an alternative to powerful antibiotics.6,7

Microbiome profiling through 16S rRNA gene sequencing has proven to be a valuable tool to characterize the diversity and composition of gut microbial communities, including in studies of CDI development and recurrence.8

Given the intricate relationship between the gut microbiota and CDI, accurate identification of C. difficile directly from 16S rRNA profiles in patient populations could be a valuable measure in future studies. However, a fundamental challenge to studying C. difficile through these approaches has been the level of taxonomic resolution provided through short 16S rRNA sequences.

As a result, most microbiome sequencing studies of CDI utilize higher aggregate taxonomic categories (e.g., the Clostridium XI cluster, which encompasses many other organisms related to C. difficile) as a proxy for the organism itself or simply avoid quantification altogether.9,10,11,12,13,14,15,16,17

Here we utilize a high-resolution method (Resphera Insight) for assigning species-level context to 16S rRNA gene sequence data to estimate C. difficile burden in different patient populations. This method was recently validated for detection of Listeria monocytogenes18 and Salmonella enterica,19,20 and was applied in this study to determine the relative abundance of C. difficile in several clinically relevant patient groups. Re-examining published 16S rRNA gene sequence datasets has confirmed previous associations of C. difficile with C. scindens, and identified new positive and negative correlations of C. difficile with other species, both of which may help provide insight into community aspects of C. difficile colonization and resistance against CDI.

Results

Evaluation of sensitivity and specificity for C. difficile identification

One of the challenges of 16S rRNA gene sequencing is the limited information available in these short DNA fragments to distinguish related microbial members below the genus-level. To accurately assess C. difficile at the species level from 16S rRNA gene sequence data, we used a method developed specifically for species level characterization (Resphera Insight, see Methods). We first validated this approach by obtaining full-length 16S rRNA gene sequences from 804 novel C. difficile isolates derived from multiple sources, and subsequently simulated noisy 16S rRNA gene sequence reads for taxonomic assignment (see Methods). Performance was measured using the Diagnostic True Positive Rate (DTP), defined as the percentage of sequences with an unambiguous assignment to C. difficile. The method achieved an average DTP of 99.9% (ranging from 98.92 to 100% per isolate, Table S1), indicating sufficient sensitivity to detect C. difficile from short 16S rRNA gene sequence reads.

In addition to establishing sufficient sensitivity to detect C. difficile, we also sought to evaluate false positive rates in which the method incorrectly assigns a sequence to C. difficile. As this species is a member of the Clostridium XI cluster, a false positive assessment was performed based on in silico simulations of 22 other members of this group, including the very similar Clostridium irregulare. Simulating 10,000 16S rRNA gene sequence reads per species with a 0.5% error rate, 20 of 22 species resulted in zero false positive assignments to C. difficile, with the highest false positive rate (0.07%) attributed to Clostridium irregulare (Table 1).

Representation of C. difficile relative to the microbiota in adult cases of CDI and healthy individuals

To examine the presence of C. difficile in different human populations, we re-examined existing published 16S rRNA gene sequencing datasets with our validated method. We first compared the relative abundance of C. difficile across a cohort of healthy individuals to two cohorts of patients diagnosed with CDI (symptomatic index cases) from Seekatz et al.10 and Khanna et al.21 (Table S2). The Seekatz protocol for CDI diagnosis followed a two-stage algorithm employing enzyme immunoassay for GDH antigen and toxins A and B, with confirmation of tcdB gene presence via PCR if toxin and GDH results were discordant; the Khanna et al. protocol for CDI diagnosis was not reported in the original publication. The healthy patient cohort and Seekatz CDI datasets were generated using equivalent processing and sequencing methods.10 Average analyzed sequencing depths per sample for CDI and healthy groups were 16,114 and 14,937, respectively.

Overall, C. difficile was detected in 58 of 70 CDI index patients (82.9%) in the Seekatz study with an average abundance of 3.04% (Fig. 1a). In the Khanna dataset, C. difficile was detected in 73 of 86 CDI index patients (84.9%) with an average abundance of 0.76% (Fig. 1b). Among healthy controls, only 18 of 211 (8.5%) harbored detectable levels of C. difficile, with an average abundance of 0.008%, significantly less than both Seekatz and Khanna index cases (P < 2e-16; Mann–Whitney test).

We were further interested in determining whether the ability to detect C. difficile or varying levels of C. difficile relative abundance from 16S rRNA gene sequences was related to disease outcome. The Seekatz dataset included samples collected from patients that went on to develop recurrent CDI, a serious outcome following primary diagnosis, or from patients who were later reinfected with CDI beyond the standard time recurrence window.10 Additionally, a severity score22 was available for some of the patients. Across the full Seekatz CDI positive sample set, our method detected C. difficile above 0.1% abundance in 59.2% of samples (Table 2). On average, patients with CDI for index (at primary diagnosis), recurrence or reinfection events had C. difficile abundances greater than 1% regardless of the calculated severity status using Infectious Diseases Society of America (IDSA) standards. We found no significant associations of C. difficile abundance with IDSA severity status among index samples or at the time of recurrence or reinfection (P > 0.05, Mann–Whitney test).

Representation of C. difficile relative to the microbiota in infants

To assess the levels of C. difficile carriage among infants relative to the total gut microbiome, we re-examined 16S rRNA gene sequence datasets describing longitudinal studies of pre-term infants in the neonatal intensive care unit (NICU) by Zhou et al.23 and a single infant profiled during the first 18 months of life by Davis et al.16 In the Zhao dataset, 12 necrotizing enterocolitis (NEC) cases and 26 age-matched controls (all treated at Brigham and Women’s Hospital NICU, Boston, MA) were sequenced with an average of seven samples per subject. The Davis asymptomatic case study consisted of profiling 50 fecal samples over time, during which researchers noted colonization switching between toxigenic and non-toxigenic strains and observed 100,000-fold fluctuations of C. difficile spore counts.16

In these two 16S rRNA gene sequence datasets, moderate levels of C. difficile (>1.0% abundance) appeared consistently within infants over time. In the Zhao dataset, C. difficile was detected in 25 of 38 (66%) infants, including 6 of 12 (50%) infants with NEC, and 19 of 26 (73%) normal infants. There was no significant difference in overall C. difficile presence between NEC and normal infants (P = 0.27, Fisher’s exact test), and both groups maintained statistically similar C. difficile abundance distributions relative to their total gut microbial communities under multivariate regression after adjustment for patient source (Fig. 2a). As the original Davis case study determined C. difficile carriage using spore counts and GDH concentration, we detected substantial representation of C. difficile (up to 7.1% abundance) until the time of weaning and transition to cow’s milk (Fig. 2b). We further found a statistically significant correlation between our C. difficile relative abundance estimates and GDH concentration measurements from the Davis study (Spearman correlation = 0.817; P = 5e-13).

Fig. 2

Distribution of C. difficile during longitudinal gut microbiome sampling of infants. a Pre-term infants in a NICU, including those developing necrotizing enterocolitis (purple) and normal (grey). Each boxplot reflects a single patient with multiple time points (total samples per patient shown along the x-axis). b A longitudinal case study of an infant before (red) and after (blue) weaning during the first 18 months of life. During the transition to cow’s milk, C. difficile relative abundance fell to undetectable levels

Correlations of C. difficile with other bacterial species

Recent studies in animal models have indicated that certain species may generate metabolites that inhibit C. difficile, such as the production of secondary bile acids by C. scindens.15 However, previous studies correlating the abundance of C. difficile with other taxa did not utilize the microbiome-based abundances directly, but rather quantified C. difficile abundance through other means such as real-time PCR, colony forming units through culture, measuring GDH concentration or spore counts.15,16,17

We sought to determine whether high-resolution analysis of the 16S rRNA gene sequence data itself could reveal the same associations, and perhaps other relevant species. Computing correlations using Compositionality Corrected by REnormalization and PErmutation (CCREPE)24 across our re-analyzed cohorts, we found a significant negative association between C. difficile and C. scindens for the Khanna CDI patient cohort and the Davis infant longitudinal study (P < 0.02 for both datasets), with a supporting trend in the other studies (Fig. 3, Table S3). Additionally, multiple members of Blautia spp. displayed a consistent negative correlation like that of C. scindens (Fig. 3, Table S3). In contrast, other Clostridia such as C. neonatale and C. paraputrificum and members of Veillonella showed strong positive associations with C. difficile abundance. In silico simulations of noisy 16S rRNA gene sequence reads from these species confirmed a low mis-assignment rate (average 0.08%; see Table S4).

Fig. 3

Correlation analysis identifies species positively or negatively associated with C. difficile. The CCREPE N-dimensional checkerboard score (y-axis) incorporates the ratio of co-variation to co-exclusion patterns normalized to a range of (−1, +1). In addition to C. scindens, we identify significant negative correlations with C. difficile for members of Blautia and positive correlations with other Clostridia and Veillonella spp. (*P ≤ 0.05). Ambiguous species level assignments are denoted by slashes. Key for re-analyzed datasets from the following studies: Recurrent CDI=10, Index CDI=21, FMT=9, Infant longitudinal=16, NICU=23 (Table S2)

Discussion

In this study, we sought to identify species-level abundances of C. difficile in 16S rRNA gene sequence datasets from different patient populations using a validated algorithm (Resphera Insight). Similar to previous studies of Listeria monocytogenes18 and Salmonella enterica,19,20 validation using a high-resolution taxonomic assignment method from 804 novel C. difficile isolates established an overall sensitivity of 99.9% with a marginal false positive rate less than 0.1%, suggesting that C. difficile could be distinguished from other related microbiota members.

Compared to the microbiota of healthy individuals, we observed a higher presence and relative abundance of C. difficile in microbiota data collected from two CDI patient cohorts. 8.5% of healthy individuals were positive for C. difficile using our approach, supporting previous epidemiological assessments of asymptomatic carriage rates.25,26,27,28 Although analysis of CDI datasets revealed a wide distribution of C. difficile relative abundances (ranging from virtually undetectable to above 50% of total sequences), the relative abundance of detected C. difficile in relation to other members of the microbiota was significantly lower in healthy individuals than that of CDI patients. The ability to assess C. difficile levels as part of the microbiota community is potentially more important within population surveys compared to diagnosis using traditional PCR or GDH/EIA tests that merely account for the presence of C. difficile using toxin B or GDH as a proxy.

While detection of C. difficile from 16S rRNA gene sequence data is limited by sequencing depth, our results suggest that C. difficile does not generally reside in healthy adults. In contrast, we did not detect C. difficile in all patients with CDI. The relative presence of C. difficile in these patients is likely below the detection limit given the available sequencing depth, however some of the samples collected from patients in the Seekatz dataset were collected during antibiotic treatment, thus potentially limiting growth of C. difficile during those time points. Indeed, Seekatz et al. report that they were unable to retrieve C. difficile strains from all patient time points via anaerobic cultivation, generally the gold standard for C. difficile detection and diagnosis.

In a third cohort of 14 recurrent CDI patients receiving fecal microbiota transplantation from nine healthy donors (FMT; Table S2, Fig. 3), C. difficile was less frequently detected than the Seekatz and Khanna index CDI patient groups. Only 4 of 14 FMT patients had any detectable levels of C. difficile before treatment, and 3 of 14 had observations of C. difficile post-FMT. Notably, Resphera Insight detected C. difficile presence in both patients who went on to develop symptomatic CDI post-FMT (recipient IDs 005 and 006).9 Prior to FMT, all patients were treated with vancomycin (125 mg 4× per day) for at least 4 days before and the day of transplantation. Thus, we attribute the reduced detection of C. difficile in this cohort to differences in patient treatment before sampling.

Applying our approach to a longitudinal dataset of 38 premature infants in a single NICU, we identified C. difficile in two-thirds of this patient cohort. Asymptomatic carriage of C. difficile among infants has been observed to be higher than for adults, and it remains unknown whether infant cases of CDI represent true disease.29,30 While CDI testing of infants is not recommended,30 recent epidemiological studies indicate 26% of children hospitalized with CDI are infants under 12 months of age, and 5% are neonates.31 In one study of 753 pediatric patients 0 to 12 years of age, 2.9% of CDI outpatients, 4.6% of CDI inpatients, and 6.6% of healthy controls were positive for C. difficile toxin B.32 Another recent study of C. difficile in 338 healthy infants (<2 yrs) in the United Kingdom found 10% were colonized at enrollment with a toxigenic strain, and 49% became colonized with a toxigenic strain post-enrollment.33 Symptomatic Clostridium difficile infections are believed not to occur in infants due to the expected lack of specific toxin receptors and under-developed signaling pathways in the gut; however, these proposed mechanisms have not been rigorously evaluated in studies of humans.34,35,36 Multiple case studies have argued that CDI can occur in this patient population,36 and there is ongoing debate about the appropriate policy for treatment of symptomatic children who test positive for C. difficile.37,38

Our analysis of an infant case study of asymptomatic colonization during the first 18 months of life identified a reduction in C. difficile relative abundance after abrupt transition from human milk to cow’s milk. Yet in a large longitudinal study by Stoesser and colleagues, multivariate analysis demonstrated that breastfeeding (mixed with formula or exclusively) was protective against asymptomatic C. difficile colonization.33 As noted by Davis and colleagues,16C. difficile does not carry the functional capacity for cleaving monosaccharides from oligosaccharide side chains and thus depends on the generation of monomeric glucose by other commensal members of the gut microbiome.39 Additionally, C. difficile relies on sialic acid as a carbon source for expansion made available by other commensals such as Bifidobacterium species.40 Therefore, the reduction of C. difficile after transition to cow’s milk is potentially the result not of milk source alone, but shifting microbial community composition and the presence of substrates by which C. difficile may thrive.

We were also able to identify a significant negative correlation between the abundance of C. difficile and C. scindens in one of the CDI cohorts, confirming similar trends reported by Buffie et al.15C. scindens, a secondary bile acid producer of deoxycholic acid which has been shown to protect against CDI, may have important translational implications.13,41 New and consistent negative correlations were also identified between C. difficile and multiple species within the Blautia genus including B. faecis, B. luti, B. schinkii, and B. wexlerae. Notably, some members of the Blautia genus are known for 7α-dehydroxylating activity of primary bile acids,42,43,44 however this remains to be evaluated for the species we identified in this study. These data suggest that species other than C. scindens may provide relevant functional capabilities in the context of CDI and prove to be informative in the development of future microbial-based therapeutics. One exception to these findings was the lack of negative correlations identified within the NICU infant cohort, which can be attributed to the very limited observations of these Blautia species and C. scindens in the overall dataset (Table S3). Indeed, among the 322 NICU infant samples analyzed, only B. luti and B. wexlerae were observed at all, and only in 5 (1.6%) and 2 (0.6%) samples, respectively, which precluded their evaluation with the CCREPE method.

While microbiome profiling through 16S rRNA gene sequencing is unlikely to replace existing methods for routine diagnosis of CDI, sequence-based assessment of C. difficile levels in the context of microbiota profiling rather than presence alone may prove valuable in surveillance of C. difficile in patient populations, prediction of disease outcome, or the development of new therapies for CDI. Although our study is limited to 16S rRNA gene-based identification of C. difficile and cannot predict whether a strain produces toxin or carries a functional pathogenicity locus,45 consideration for accurate identification of C. difficile and related members may be useful in assessing clinical outcomes of new microbial therapies that rely on 16S rRNA gene sequencing to validate recovery of the microbiota.

Methods

Validation of Resphera Insight for identification of C. difficile

Whole-genome shotgun sequence datasets available from (i) The Wellcome Trust Sanger Institute and (ii) The University of Maryland Institute for Genome Sciences designated as novel C. difficile isolates were downloaded from the NCBI Sequence Read Archive (see Table S1 for accessions), trimmed for quality using Trimmomatic46 and assembled into contigs using Minia.47 Contigs containing portions of 16S rRNA genes were identified using BLASTN48 and extracted for amplicon simulations. For each isolate, we subsequently simulated 16S rRNA amplicon sequence reads (10,000 per isolate) from the V4 region (the primary amplicon region selected in the real datasets) with a random nucleotide error rate of 0.5%. The Diagnostic True Positive Rate was computed as the percentage of sequences unambiguously assigned by Resphera Insight to C. difficile.

Processing of 16S rRNA gene sequence datasets

Raw 16S rRNA gene sequence datasets were processed as follows: Raw overlapping paired-end reads were merged into consensus fragments by FLASH49 requiring a minimum 20 bp overlap with 5% maximum mismatch density, and subsequently filtered for quality (targeting error rates < 1%) and length (minimum 200 bp) using Trimmomatic46 and QIIME.50 Spurious hits to the PhiX control genome were identified using BLASTN and removed. Sequences were then trimmed of their associated primers, evaluated for chimeras with UCLUST (de novo mode),51 and screened for human-associated contaminants using Bowtie252 searches of NCBI Homo sapiens Annotation Release 106. Mitochondrial contaminants were detected and filtered using the RDP classifier53 with a confidence threshold of 50%, and passing high-quality 16S rRNA gene sequences were subsequently assigned to a high-resolution taxonomic lineage using Resphera Insight (Baltimore, MD).18,19,20,54,55 Briefly, the method relies on (i) a manually curated 16S rRNA gene database including 11,000 unique species and (ii) a hybrid global-local alignment strategy to assign sequences a species-level taxonomic lineage. While the method attempts to achieve species-level resolution, if the internal statistical model indicates uncertainty in final species membership, the tool minimizes false positives by providing “ambiguous assignments” i.e., a list of species reflecting all relevant candidates. For example, if a 16S rRNA gene fragment is ambiguous between Veillonella atypica and Veillonella dispar, the algorithm will provide the ambiguous assignment: “Veillonella_atypica:Veillonella_dispar.”

Statistical analyses

Correlations between C. difficile and other species were computed using CCREPE (v.1.10.0)24 (http://huttenhower.sph.harvard.edu/ccrepe). CCREPE (Compositionality Corrected by REnormalization and PErmutation) utilizes an N-dimensional extension of the checkerboard score particularly suited to similarity score calculations between compositions derived from ecological relative abundance measurements of co-occurrence or co-exclusion. Two sample statistical comparisons utilized the Mann-Whitney U test unless otherwise noted.

In silico evaluation for species identified in CCREPE analysis

For single species reported in CCREPE correlation analysis, we simulated noisy 16S rRNA gene sequences (V4 region; 0.5% error rate; 1000 seqs per species), and calculated the frequency of (1) assignments that included the correct species (allowing for ambiguous assignments), (2) unambiguous assignments to the correct species, and (iii) mis-assignments that did not include the correct species (Table S4).

Ethics approvals and consent to participate

IRB approval and patient consent statements from each study: Recurrent CDI (Seekatz et al.10)—All subjects signed written consent to participate in this study. This study was approved by the University of Michigan Institutional Review Board (Study HUM33286; originally approved 8/26/2009).

NICU Infants (Zhou et al.23)—Samples were collected following a protocol that was approved by the Partner’ s Human Research Committee (IRB) for Brigham and Women’ s Hospital. All study procedures were approved by the IRBs at both Brigham and Women’ s Hospital in Boston, MA and at The Genome Institute in St. Louis, MO. The IRB deemed this study to be of minimal risk with no interaction and no intervention with human subjects and thus, was exempt from consent.

Infant Longitudinal (Davis et al.16)—The study was approved by the TechLab Institutional Review Board and included informed consent obtained from the mother.

Healthy Controls (Seekatz et al. submitted)—All subjects signed written consent to participate in this study. This study was approved by the University of Michigan Institutional Review Board (Study HUM33286; originally approved 8/26/2009).

Acknowledgements

We thank Cynthia Sears, Karen Carroll, and David Cook for helpful suggestions on this work. This work was supported in part by the ERIN CRC (Enteric Research Investigative Network Cooperative Research Center), (U19AI09087, NIAID), awarded to V.B.Y. A.M.S. supported by the National Center for Advancing Translational Sciences (UL1TR000433).

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A tapered and pulsed regimen with vancomycin — with diligent follow-up — can achieve significant cure rates in recurrent Clostridium difficile (C. difficile) infected patients, according to a new study.

Researchers from Loyola Medicine retrospectively studied 100 vancomycin taper and pulse treatment patients treated for recurrent C. difficile infection between January 1, 2009 and December 31, 2014. Their clinic, the study authors wrote, has been a referral center for the infection for the past decade.

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However, despite the guidelines for treatment of recurrent C. difficile infection being not too different than recurrent episodes – except for the use of vancomycin when the case is severe – there have not been many studies on this vancomycin taper and pulsed dosing.

The researchers observed that after a referral, the confirmed recurrent C. difficile patients were treated with a vancomycin taper and pulse regimen: a taper of vancomycin to once-daily, followed by alternate day dosing; or once-daily followed by alternate day dosing; followed by every third day, for at least 2 weeks. After this regimen, all patients had 90-day follow-up documentation.

On average, the patients in the clinic were on their third C. difficile diarrhea episode. Half of the patients had also received a standard course of vancomycin, while another third had received some type of vancomycin taper regimen, the researchers said.

Despite the fact that many of these patients were a “treatment experienced” population, 75% of the patients who received a supervised vancomycin taper and pulsed regimen achieved a cure, study author Stuart Johnson MD, . He added that the results were further improved for patients who received the expended pulse phase: 81% achieved a cure.

“The findings were not unexpected to us, but I think that many clinicians will be surprised how well a deliberate, prolonged vancomycin taper and pulse regimen – with careful follow up – works,” Johnson said.

There were no significant differences among the patients in terms of gender, age, concomitant antibiotics, proton pump inhibitor use, histamine receptor-2 blocker use, or patients with a regimen greater than 10 weeks in length, the researchers continued.

The researchers added that their finding of improved cure rates with alternate-day dosing plus every third day dosing over strictly alternate-day dosing is consistent with the hypothesis that pulsed dosing can promote a cyclical decrease in spore burden, they wrote. This can also permit the resetting of normal microbiota in the gut.

Johnson concluded that the clinical implications of the study show most recurrent C. difficile patients do not need fecal microbiota transplant (FMT).

“FMT has received an enormous amount of press and this procedure is now widely available throughout the US,” Johnson said. “FMT is attractive because it addresses one of the primary mechanisms involved with recurrent C. difficile infection, a marked disruption of the resident bacteria that populate the intestine and provide an important host defense against C. difficile.

Although physicians screen donor feces for “known pathogens,” not all is known of the potential complications to come from FMT, Johnson said.

“In addition, it appears that efficacy with a carefully supervised vancomycin taper and pulse regimen compare to that achieved with FMT,” Johnson said.

New research shows, it can’t make this last, crucial move without enough of a humble nutrient: calcium.

And that new knowledge about Clostridium difficile (a bacterium also known as “C. diff“) may lead to better treatment for the most vulnerable patients.

The discovery, made in research laboratories at the University of Michigan Medical School and the U.S. Food and Drug Administration, is published in the online journal PLoS Pathogens.

It helps solve a key mystery about C. diff: What triggers it to germinate, or break its dormancy, from its hard spore form when it reaches the gut.

Though the findings were made in mice, not humans, the researchers say the crucial role of calcium may help explain another mystery: Why some hospital patients and nursing home residents have a much higher risk of contracting C. diff infections and the resulting diarrhea that carries its spores out of the body.

That group includes people whose guts are flooded with extra calcium because they’re taking certain medications or supplements, have low levels of Vitamin D in their blood or have gut diseases that keep them from absorbing calcium.

The new discovery shows that C. diff can recognize this extra calcium, along with a substance called bile salt produced in the liver, to trigger its awakening and the breaking of its shell.

Previous research had suggested it couldn’t do this without another key component, an amino acid called glycine. But the new findings show calcium and the bile salt called taurochlorate alone are enough. Mouse gut contents that were depleted of gut calcium had a 90 percent lower rate of C. diff spore germination.

“These spores are like armored seeds, and they can pass through the gut’s acidic environment intact,” says Philip Hanna, Ph.D., senior author of the new paper and a professor of microbiology and immunology at U-M. “Much of the spore’s own weight is made of calcium, but we’ve shown that calcium from the gut can work with bile salts to trigger the enzyme needed to activate the spore and start the germination process.”

Ironically, the researchers say, one way to use this new knowledge in human patients might be to add even more calcium to the system.

That could awaken all the dormant C. diff spores in a patient’s gut at once, and make them vulnerable to antibiotics that can only kill the germinated form. That could also prevent the transmission of more spores through diarrhea to the patient’s room. That could slow or stop the cycle of transmission that could threaten them or other patients in the future.

Hanna’s graduate student, Travis Kochan, made a key observation that led to the discovery. He noted that the fluid “growth medium” that the researchers typically grow C. diff in for their studies had calcium in it. He realized this could artificially alter the results of their experiments about what caused C. diff spores to germinate.

So, he used a chemical to remove the calcium while leaving all the other nutrients that keep C. diff growing. The result: no new spore germination happened in the calcium-free growth medium.

FDA’s Center for Biologics Evaluation and Research conducted further research in laboratory dishes and in the guts of mice. FDA’s Paul Carlson, Ph.D., a former U-M research fellow, and fellow FDA scientists in his laboratory found that C. diff spores that were mutated so that glycine couldn’t act on them could still germinate and colonize mice. This suggested that calcium, and not glycine, was critical for this process.

Both mutant and regular forms of the bacteria could still activate an enzyme inside the C. diff spore that led the bacteria to start dissolving their hard shell. This released the store of calcium that the spore had been harboring inside itself, and increases the local level of the nutrient even further.

“These spores don’t want to germinate in the wrong place,” says Kochan, whose grandfather suffered from a severe C. diff infection which ultimately led to his death. “C. diff spores have specialized to germinate in the gut environment, especially in the environment of the small intestine, where calcium and the bile salt injection from the liver comes in.”

Hanna notes that the bile salt connection to C. diff spore germination was first discovered at U-M in 1982 by a team led by Ken Wilson, M.D.

Calcium and the gut

Certain ailments and treatments cause defects in calcium absorption, but are also risk factors for C. diff infections. For example, patients with vitamin D deficiency are five times more likely to get C. diff.

Medications aimed at calming acid reflux – such as proton pump inhibitors – and steroids can increase the amount of calcium in the gut. A Vitamin D deficiency can keep the body from reabsorbing calcium through the gut wall, allowing it to build up.

And people with inflammatory bowel diseases such as Crohn’s and colitis also have a harder time absorbing calcium from food through their gut walls.

Older adults are also often counseled to take calcium supplements to compensate for lower calcium levels and protect their bones from fracturing.

Hanna cautions that the new findings should not cause any patients to stop taking their medications or doctor-recommended supplements, or to start taking new ones. But he hopes to work with clinicians at U-M and beyond to test the new knowledge in a clinical setting. Meanwhile, he and Kochan and their FDA and U-M colleagues will continue to study C. diff germination in mice and look for ways to block the enzymes crucial to spore germination.

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As part of a multicenter study, investigators from the University of Oxford, the University of Leeds, Astellas Pharma Europe, and elsewhere used a combination of ribotyping, sequencing, phylogenetics, and geographic analyses to retrace the genetic diversity and potential sources of C. difficile isolates involved in infections in European hospitals.

Recent research suggests a proportion of Clostridium difficile cases in Europe involve not only hospital-acquired infections but also infections associated with other sources, such as food.

David Eyre, a clinical lecturer at the University of Oxford, was slated to present the work at the European Congress of Clinical Microbiology and Infectious Diseases annual 2017 meeting in Vienna this past weekend. The study was funded by Astellas Pharma’s Europe, Middle East, and Africa (EMEA) program.

“We don’t know much about how C. difficile might be spread in the food chain, but this research suggests it may be very widespread,” Eyre said in a statement. “If that turns out to be the case, then we need to focus on some new preventative strategies such as vaccination in humans once this is possible, or we might need to look at our use of animal fertilizers on crops.”

“This study doesn’t give us any definitive answers,” he explained, “but it does suggest other factors [than hospital infections] are at play in the spread of C. difficile and more research is urgently needed to pin them down.”

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Some of the strains clustered by locale, consistent with spread from one individual to the next, for example in a healthcare setting. But more unexpectedly, the team also saw strains smattered across seemingly unconnected sites. And because at least one of those strains had previously been linked to pig farming, the researchers speculated that some infections may have been transmitted through food sources.

Abstract

Background

Clostridium difficile is a nosocomial pathogen prevalent in hospitals worldwide and increasingly common in the community.

Sequence differences have been shown to be present in the Surface Layer Proteins (SLPs) from different C. difficileribotypes (RT) however whether these differences influence severity of infection is still not clear.

Results

We used a molecular evolutionary approach to analyse SLPs from twenty-sixC. difficile RTs representing different slpA sequences. We demonstrate that SLPs from RT 027 and 078 exhibit evidence of positive selection (PS).

We compared the effect of these SLPs to those purified from RT 001 and 014, which did not exhibit PS, and demonstrate that the presence of sites under positive selection correlates with ability to activate macrophages.

SLPs from RTs 027 and 078 induced a more potent response in macrophages, with increased levels of IL-6, IL-12p40, IL-10, MIP-1α, MIP-2 production relative to RT 001 and 014. Furthermore, RTs 027 and 078 induced higher expression of CD40, CD80 and MHC II on macrophages with decreased ability to phagocytose relative to LPS.

Conclusions

These results tightly link sequence differences in C. difficile SLPs to disease susceptibility and severity, and suggest that positively selected sites in the SLPs may play a role in driving the emergence of hyper-virulent strains.

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